FAK is a tyrosine kinase that functions in regulating cell shape, motility and survival. FAK may play a role in the development of human cancer, since FAK is overexpressed in some tumors and artificial enhancement of FAK signaling can promote some tumorigenic properties in cells. The C-terminal domain of FAK, called the FAT sequence, associates with other proteins like paxillin, and functions in localizing FAK to cellular focal adhesions. Paxillin has also been implicated in the control of cell shape and motility, and thus the FAK/paxillin complex may be important for these functions. The FAT sequence is critical for the regulation of FAK and for the transmission of signals downstream of FAK suggesting that this domain might be a future therapeutic target for inhibition of FAK. Recent structural studies have revealed that the FAT sequence is an anti-parallel 4-helix bundle, although a second structure suggests that this domain can form a domain swapped dimer. This finding suggests that there may be dynamics in helix 1, the domain that is swapped in the dimer. Further, two models of paxillin binding to FAK have been proposed, a two-binding site model and a one-binding site model. Thus, there are a number of outstanding structural questions that have yet to be addressed. This proposal describes a combined structural and biochemical/molecular biology approach to the analysis of the FAT sequence of FAK. The solution Istructure of the FAT sequence will be solved by NMR with an emphasis on the analysis of the dynamics of the hinge between helices 1 and 2. Mutations in the hinge will be engineered and characterized to assess the role of hinge dynamics in the function of FAK. To further explore the structural basis of the FAK/paxillin interaction, the solution structure of the FAT sequence in complex with a synthetic peptide mimicking one FAK-binding site of paxillin will be solved by NMR. A series of experiments will be performed to test the hypothesis that the FAK/paxillin complex plays an important role in regulating cell spreading and motility. These experiments will include characterization of paxillin mutants defective for FAK binding to determine the role of this interaction in paxillin function and characterization of FAK mutants that are compromised for association with paxillin to determine the role of paxillin binding in FAK function.